Pathological and phylogenetic features of prevalent canine distemper viruses in wild masked palm civets in Japan

Ten wild masked palm civets infected with canine distemper virus (CDV), captured in Japan from 2005 to 2007, were histopathologically and phylogenetically analyzed. Phylogenetic analysis based on the amino acid sequences of the H protein of two CDV isolates from masked palm civets revealed that the two isolates were classified into the clade of recent isolates in Japan. Histopathologically marked lesions of virus encephalitis were present in the brain, whereas gastrointestinal lesions were absent or at a mild degree. The distribution of the lesions resembles that of recent CDV cases in dogs. Therefore, recent CDV infections in masked palm civets could be caused by recently prevalent CDV in dogs. The possibility of the masked palm civet as a spreader of CDV among wildlife is also discussed.     

Prevalence of Salmonella, Yersinia and Campylobacter spp. in feral raccoons (Procyon lotor) and masked palm civets (Paguma larvata) in Japan

To estimate the public and animal health risk that alien species pose, the prevalence of Salmonella, Yersinia, and Campylobacter spp. in feral raccoons (Procyon lotor, n=459) and masked palm civets (Paguma larvata, n=153), which are abundant alien species in Japan, was investigated in urban and suburban areas of Japan. Salmonella enterica was detected from 29 samples [26 raccoons, 5.7%, 95% confidence interval (CI) 7.8-3.5%; three masked palm civets, 2.0%, 95% CI 4.2-0%]. Many of the isolates belonged to serovars that are commonly isolated from human gastroenteritis patients (e.g. S. Infantis, S. Typhimurium, and S. Thompson). The antimicrobial susceptibility test showed that 26.9 % of the isolates from raccoons were resistant to at least one antimicrobial agent, whereas none of the isolates from masked palm civets were resistant. Yersinia sp. was detected from 193 samples (177 raccoons, 38.6%, 95% CI 43.0-34.1%; 16 masked palm civets, 10.5%, 95% CI 15.3-5.6%). All virulent Yersinia strains belonged to Yersinia pseudotuberculosis, which was isolated from seven (1.5%, 95% CI 2.6-0.4%) raccoons and six (3.9%, 95% CI 7.0-0.8%) masked palm civets. According to the detection of virulence factors, all the Y. pseudotuberculosis isolates belonged to the Far Eastern systemic pathogenicity type. Campylobacter spp. was detected from 17 samples (six raccoons, 1.3%, 95% CI 2.3-0.3%; 11 masked palm civets, 7.2%, 95% CI 11.3-3.1%). Among these, three isolates from raccoons were identified as C. jejuni. These results showed that these pathogens can be transmitted by human activities, other wild animals, and the environment to feral raccoons and masked palm civets, and vice versa. As these animals have omnivorous behaviour and a wide range of habitats, they can play an important role in the transmission of the enteric pathogens.     

Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy

Canine distemper virus (CDV) is a highly contagious viral pathogen causing lethal disease in dogs and other mammalians. A high degree of genetic variation is found between recent CDV strains and the old CDV isolates used in the vaccines and such genetic variation is regarded as a possible cause of the increasing number of CDV-related diseases in dogs. The H gene shows the greatest extent of genetic variation that allows for distinction of various lineages, according to a geographical pattern of distribution and irrespective of the species of identification. In the present study, hemagglutinin (H) genes obtained from field strains detected from clinical specimens of Italian dogs were analyzed genetically. Phylogenetic analysis revealed that a homogeneous group of CDV strains is widespread in Italian dogs, all which are included into the European lineage. Unexpectedly, strains 179/04 and 48/05 clustered along with CDVs of the Arctic lineage, the highest identity being to strain GR88 (98.0 and 98.4%aa, respectively). The full-length sequence of a red fox CDV strain, 207/00 was also determined and analyzed. The H protein of the fox CDV strain was unrelated to strains within the major European lineage. These results suggest that at least three different CDV lineages are present in Italy.     

犬瘟热病毒H基因的序列分析与表达

以临床疑似犬瘟热病犬外周血总RNA为模板,采用RT-PCR方法扩增犬瘟热病毒(CDV)血凝素(H)基因。序列测定和分析表明该病料犬瘟热病毒H基因序列与国内其他地区分离到的毒株同源性较高(94.6%~99.2%),而与Onderstepoort株、Convac株等疫苗毒株的同源性较低(90.4%~91.2%)。分子遗传进化分析显示所有毒株可分为7个大的分支,且各分支间具有一定的地域性,其中待检病料中CDV与大多数中国分离株一样处于Ⅰ型。进一步以H全基因为模板,截短表达其3′端816 bp、459 bp两个片段,并克隆入原核表达载体pET30 a进行表达。结果显示它们分别能表达大小约为36.4 ku和22.2 ku的融合蛋白。免疫转印表明该纯化蛋白均可与犬瘟热病毒抗血清发生阳性反应,说明重组蛋白具备抗原性,可用于进一步的血清学研究。     

基于H蛋白基因的犬瘟热病毒遗传变异分析

犬瘟热是犬瘟热病毒(Canine distemper virus,CDV)感染犬和其他食肉动物造成的多发性、致死性传染病,本文从分子水平上探讨CDV遗传进化特性、变异情况与流行规律之间的关系.通过收集2002-2010年在中国地区分离的14株CDV野毒株、2006-2007年在全球各地分离的12株CDV野毒株以及从不同宿主分离的12株CDV野毒株和4株疫苗株,将其分为4组,将前3组野毒株分别与国内外正在使用的4株疫苗株的H基因进行遗传变异分析.分析发现,CDV野毒株与疫苗株间H蛋白基因的核苷酸相似性为86.2％～92.1％,其氨基酸相似性为89.1％～91.9％;H基因的584位的天冬酰胺糖基化位点是Asia-Ⅰ型CDV所特有的;H蛋白3555区域的非同义氨基酸替换概率较高.作者推测H蛋白抗原变异可能造成弱毒疫苗免疫效力降低,不能为某些CDV株的感染提供完全有效的保护.     

水貂、狐狸和貉源犬瘟热病毒F蛋白信号肽区基因克隆及遗传变异分析

为了解近年来犬瘟热病毒(canine distemper virus,CDV)在中国毛皮动物主要养殖区的流行情况及遗传变异情况,本试验于2012-2014年从山东、河北、辽宁收集疑似犬瘟热的水貂、狐狸和貉病料,经犬瘟热抗原检测试纸条检测和RT-PCR检测为阳性后,克隆测序了15株CDV F蛋白信号区(Fsp)基因序列.序列分析结果显示,15株CDV野毒株Fsp基因核苷酸序列和氨基酸序列的相似性均为93.6%~100.0%,与疫苗株相似性为80.7%~81.7%.基因系统进化分析结果显示,15株野毒株均属于中国当前流行的Asia-1基因型.氨基酸序列比对分析结果显示,Fsp蛋白在氨基酸3-14、16-37、47-67位等处存在高变异.通过对当前流行CDV野毒株Fsp基因序列分析,结果表明该Fsp基因区具有较高的变异率,可作为CDV基因分型的依据,同时本试验结果为毛皮动物犬瘟热的防控提供了参考依据.     

鉴别犬瘟热病毒疫苗株和野毒株RT—PCR—RFLP方法的建立及应用

根据Onderstepoort株H基因序列,设计1对引物建立RT-PCR-RFLP检测方法,对不同宿主来源的疑似犬瘟热临床样品进行检测,并对检出的犬瘟热病毒野毒山东株PCR产物进行克隆和序列分析,验证RT—PCR—RFLP检测方法。结果RT—PCR扩增片段为1921bp,产物经RFLP分析,野毒株的PCR产物能被NdeI酶切为1282、345和294bp3个片段,弱毒疫苗株则不能被切开;病毒RNA的最小检出量为2．15ng。临床检测共检出19份样品为犬瘟热阳性,其中15份为野毒株感染,其H基因编码区全长为1824bp,均在1279和1543处有NdeI酶切位点,推导氨基酸与野毒株的同源性在92．9％～96．9％,与疫苗株的同源性在89．0％～90．8％,进化树分析显示这些毒株在基因型上属于Asia-1型,为野毒株谱系。该方法的建立为临床上犬瘟热病毒的鉴别检测和诊断提供了依据。     

Sequence analysis of the genes encoding the phosphoprotein of recent isolates of canine distemper virus in Japan

The nucleotide sequences of the phosphoprotein (P) of canine distemper virus (CDV) strains isolated between 1992 and 1996 in Japan were determined. This is the first report of the complete sequences of the P genes of recently prevalent CDV strains. The deduced amino acid sequences of the P, C and V proteins showed that in the new Japanese isolates, these proteins have approximately 93%, 90-91% and 92% identities with those of the Onderstepoort vaccine strain, respectively. The predicted functional regions were conserved. RNA editing resulting in a shift to the open reading frame (ORF) of the V protein was shown to occur with the same efficiency in both the field isolates and vaccine strain.     

Epidemiology of sarcoptic mange in free-ranging raccoon dogs (Nyctereutes procyonoides) in Yokohama,Japan

Free-ranging raccoon dogs (Nyctereutes procyonoides) from Nogeyama Zoological Gardens, Kanazawa Zoological Gardens, and Yokohama Zoological Gardens frequently rescued dogs having Sarcoptes scabiei infestation. However, the epidemiology of S. scabiei infestation has not yet been elucidated. In the present study, we investigated the epidemiology of S. scabiei infestation in raccoon dogs and its influence on the population of masked palm civets in Yokohama, Japan. We examined records of raccoon dog rescue between 1981 and 2010 and classified the dogs into the following 4 categories on the basis of the reason for rescue: dogs with S. scabiei infestation, scabies-infested dogs involved in car accidents, uninfested dogs involved in car accidents, and other reasons for rescue. We found that the number of dogs rescued due to car accidents and other reasons increased from 1989 onwards, and an S. scabiei outbreak was recorded since 1993. The infestation spread from the southern to the northern regions of Yokohama. The total number of raccoon dogs rescued annually peaked in 1995 and declined thereafter. The number of masked palm civets (Paguma larvata) rescued gradually increased with a decline in the number of raccoon dogs rescued. In the present study, we revealed the epidemiology of S. scabiei infestation in the raccoon dog. The outbreak might be induced by the increased population density, and the infestation spread immediately from the southern to the northern regions of Yokohama since 1993. Further, the population of masked palm civets may have increased due to the decrease in the population of the raccoon dog.     

Analysis of the H gene, the central untranslated region and the proximal coding part of the F gene of wild-type and vaccine canine distemper viruses.

This paper summarizes the results of the genetic analysis of several parts of the genome of canine distemper virus (CDV) field isolates and vaccine viruses. The haemagglutinin (H) gene analysis showed that recent viruses did not differ significantly from vaccine strains. The analysis of the long untranslated region between the matrix (M) and fusion (F) gene revealed distinct genetic heterogeneity. The putative F protein start codon AUG461 of vaccine strain Onderstepoort was found to be mutated in all wild-type isolates and in another vaccine strain. The proximal coding part of the F gene was well conserved. Phylogenetic analysis of this segment showed the presence of several cocirculating CDV genotypes.     

Susceptibility of carnivore hosts to strains of canine distemper virus from distinct genetic lineages

Using the complete haemagglutinin (HA) gene and partial phosphoprotein (P) gene we investigated the genotype of canine distemper virus (CDV) strains recovered from two wildlife species in Mecklenburg-Vorpommern, Germany. Phylogenetic analyses demonstrated significant differences between the strains from raccoons Procyon lotor (family Procyonidae) obtained in 2007 and strains from red foxes Vulpes vulpes (family Canidae) obtained in 2008. The raccoon strains belonged to the CDV European wildlife lineage whereas the red fox strains belonged to the CDV Europe lineage. We combined our genetic sequence data with published data from 138 CDV stains worldwide to investigate the proposed importance of amino acid substitutions in the SLAM binding region of the CDV HA protein at position 530 (G/E to R/D/N) and 549 (Y to H) to the spread of domestic dog-adapted CDV strains to other carnivores. We found no evidence that amino acid 530 was strongly affected by host species. Rather, site 530 was conserved within CDV lineages, regardless of host species. Contrary to expectation, strains from non-dog hosts did not exhibit a bias towards the predicted substitution Y549H. Wild canid hosts were more frequently infected by strains with 549Y, a pattern similar to domestic dogs. Non-canid strains showed no significant bias towards either H or Y at site 549, although there was a trend towards 549H. Significant differences between the prevalence of 549Y and 549H in wild canid strains and non-canid strains suggests a degree of virus adaptation to these categories of host.     

Notoedric mange in free-ranging masked palm civets (Paguma larvata) in Japan

Fifty-one masked palm civets (Paguma larvata) were trapped as part of a nuisance wildlife control programme between July 2001 and August 2002 in Fujisawa City, Kanagawa Prefecture, Japan. Eleven civets had characteristic mange lesions with marked alopecia and crusts, caused by the burrowing epidermal mite Notoedres cati. The diagnosis was confirmed by histology and examination of mites obtained from skin scrapings of affected civets. Histopathology of lesions demonstrated moderate hyperplastic epidermis, parakeratosis and acanthosis. Occasionally microvascular angiogenesis was observed in the epidermis. Tunnels were excavated in the hair follicles, reaching the hair roots. The tunnels were located between the hair shaft and the inner root sheath. Female mites, eggs and nymphs were demonstrated in their tunnels using scanning electron microscopy.     

Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from breeding foxes,raccoon dogs and minks in China

Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. Genetic/antigenic heterogeneity has been observed among the various CDV strains, notably in the haemagglutinin (H) gene, that appears as a good target to gather epidemiological information. Based on sequence analysis of the H gene, wild-type CDV strains cluster into distinct geographic lineages (genotypes), irrespective of the species of isolation. The sequence of the H gene of 28 CDV strains detected from both vaccinated and non-vaccinated breeding foxes, raccoon dogs and minks from different geographical areas of China during the years 2004–2008 was determined. All the CDV strains but two (strains HL and HLJ2) were characterized as Asia-1 genotype and were highly similar to each other (96.2–99.7% at the amino acid [aa] level) and to other Asia-1 strains (96.1–99.5% aa) previously detected in China. The CDV strains HL and HLJ2 were both collected from foxes in Heilongjiang province in 2005. Strain HL resembled CDVs of the Arctic genotype (GR88-like) and displayed high aa identity (98.0%) to the Chinese canine strain Liu. By converse, strain HLJ2 was barely related to CDVs of the Asia-2 genotype (88.7–90.3% aa identity), and could represent a novel CDV genotype, tentatively proposed as Asia-3. These results suggest that at least three different CDV genotypes, distantly related (81.8–91.6% aa identity) to the vaccine strains, Onderstepoort-like (America-1 genotype), are currently circulating in breeding foxes, raccoon dogs and minks in China, and that the genotype Asia-1 is predominant. Whether the diversity between wild-type CDVs and the vaccine strains may affect, to some extent, the efficacy of the vaccines deserves further investigations.     

Identification of a genetic variant of canine distemper virus from clinical cases in two vaccinated dogs in Mexico

Canine distemper virus (CDV) is a highly contagious viral pathogen of worldwide distribution that can cause lethal disease in dogs and other mammals. Genetic diversity is found among reference strains and isolates of CDV, mainly in the haemagglutinin protein (H), fusion protein (F) and nucleoprotein (N), and this may be associated with the increasing incidence of distemper in dogs. CDV was identified by RT-PCR in serum samples taken from two clinically diseased, previously vaccinated Mexican dogs. Subsequently, in both samples, a fragment of the CDV N gene was sequenced revealing a 100% identity between nucleotide sequences. However, the sequence obtained was different to that found in virus strains used in vaccines and in isolates reported elsewhere, but was closely related to A75/17, 1127/Gi95, and 2495/Gi95 sequences from USA and Germany, and clustered with 1127/Gi95 and 2495/Gi95 strains. The results suggest that a novel CDV lineage may be present in Mexico.     

Propagation of Asian isolates of canine distemper virus (CDV) in hamster cell lines

Backgrounds

The aim of this study was to confirm the propagation of various canine distemper viruses (CDV) in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance.

Methods

The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST) cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains) and by observing the development of cytopathic effect (CPE) in infected cultures of hamster cell lines.

Results

Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively.

Conclusion

The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.     

Antigenic differences in the H proteins of canine distemper viruses

Antigenic properties between new Japanese field isolates and vaccine strains of canine distemper virus (CDV) have been compared using four monoclonal antibodies (MAbs) (JD-5, JD-7, JD-11 and d-7) against the hemagglutinin (H) proteins of CDV. JD-5, JD-7 and JD-11 are newly established antibodies. Three MAbs, namely d-7, JD-5 and JD-11, reacted similarly to all the CDV strains examined. However, JD-7 reacted much more strongly with the vaccine strains and an old field isolate than the recent field isolates in immunofluorescence, radio immunoprecipitation and virus neutralization assays. These results indicate that an antigenic region in the H protein, concerned with neutralization and recognized by JD-7, has been altered in the recent field isolates.     

Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina

RT-PCR was used to detect canine distemper virus (CDV) RNA in clotted blood from Argentine domestic dogs. The NP gene was detected in 73 out of 99 blood samples analyzed. The deduced amino acid sequence of these gene fragments showed 100% identity with the sequence of other wild-type and vaccine strains. A fragment of the hemagglutinin gene was amplified from 24 (32.9%) of the NP-RNA-positive clinical specimens. These H fragments were further analyzed by restriction fragment length polymorphism (RFLP) and sequencing. A single NdeI site was detected in all 24 wild-type strains but was absent in the vaccine strains. Phylogenetic analysis of the partial hemagglutinin amino acid sequences showed close clustering for local strains, clearly distinct from vaccine strains and other wild-type foreign CDV strains. One of the local strains, Arg 23, branched out of the root of the Argentine clade, close to the European strains, suggesting that two different pathogenic CDV genotypes are currently circulating in Argentina, one of them clearly predominant.     

貉源犬瘟热病毒血凝素基因的克隆及序列分析

为了对1例貉源犬瘟热(CD)进行病毒检测并分析其血凝素H基因变异情况,本研究从1只疑似犬瘟热病死的貉采病料进行研磨,利用RT-PCR方法扩增犬瘟热病毒H基因,对扩增出的H基因片段进行克隆测序,并对得到的H基因序列进行分析。结果表明,该貉感染犬瘟热病毒,得到的H基因核苷酸和氨基酸序列与CDV野毒株同源性较高,分别为89.1%~98.0%和85.4%~97.5%。遗传进化分析表明其属于Asia-1型野毒株,N连接糖基化位点分析结果表明,该毒株在542aa处比参考野毒株多了1个潜在的N糖基化位点,在525aa-550aa间多出了1个抗原表位。     

果子狸源细小病毒分离鉴定及VP2基因遗传进化分析

【目的】探究果子狸源细小病毒流行特点及其VP2基因遗传变异情况,为细小病毒防治提供理论依据和技术支撑。【方法】采用猫肾细胞(CRFK细胞)对患有肠炎的果子狸肠道组织样品进行病毒分离,通过血凝试验、免疫荧光试验鉴定分离的病毒,并对病毒VP2基因进行PCR扩增和遗传进化分析。【结果】分离到的毒株在CRFK细胞中培养出现细胞脱落、崩解和破碎等典型细小病毒细胞病变效应(CPE);血凝试验结果显示,此病毒可凝集猪红细胞,血凝效价为1∶512;免疫荧光试验结果显示,在接种分离毒株的CRFK细胞内可观察到亮绿色荧光,而对照细胞没有荧光。将分离毒株命名为MPCPV-SX。VP2基因进化树分析发现,其与犬细小病毒处于同一分支,位于CPV-2和CPV-2a型之间,同时VP2氨基酸的87和101位显示为CPV-2型,而300和305位氨基酸则与CPV-2a型一致。【结论】本研究成功从果子狸肠道组织样品中分离出1株细小病毒MPCPV-SX,其VP2基因是介于CPV-2和CPV-2a之间的中间型,表明细小病毒通过果子狸等野生动物跨越宿主流行传播,可能在细小病毒遗传进化方面起到一定的过渡作用。     

小熊猫源犬瘟热病毒株H、F基因的克隆及序列分析

为了解1株圈养小熊猫源犬瘟热病毒（CDV）GD-1的遗传变异情况,通过RT-PCR方法对该株CDV进行H、F基因的克隆、测序及序列分析。结果显示：该分离株的H基因序列与GenBank中丹麦报道的登录号为GU266280的犬源CDV毒株的核苷酸序列相似性最高,为96%;F基因序列与巴西报道的登录号为KY057355的犬源CDV的核苷酸序列相似性最高,为95.7%。下载CDV代表毒株序列进行遗传演化、氨基酸序列比对及分子特征分析。结果显示：H蛋白共有8个潜在的N-糖基化位点,分别位于19、149、309、391、422、456、587、603位点;H蛋白的SLAM受体结合位点氨基酸序列与欧亚野生型毒株一致,与疫苗株相比,530、549位氨基酸不同,与其他CDV参考毒株H蛋白相比还存在24、41等9处氨基酸位点发生明显变异,与标准强毒株A75/17的氨基酸相似性为95.2%,与Onderstepoort、Convac等5株疫苗株的氨基酸序列相似性为88.2%~89.3%;F蛋白共有6个N-糖基化位点,分别位于62、108、141、173、179、517位,与Onderstepoort等疫苗株氨基酸相似性为89.1%~89.7%;与其他参考毒株相比还存在115、130等11处氨基酸发生变异;构建基于H、F基因的遗传进化树,结果显示：该毒株位于Asia-4型的一个小的进化分支,这与目前我国流行毒株主要位于Asia-1型存在明显不同。本研究首次报道了小熊猫源的Asia-4基因型CDV野毒株,并对毒株的H及F基因进行了序列分析,对于了解我国CDV流行株的遗传变异情况、流行病学调查、疾病防控及疫苗研发等具有重要意义。